PROJECT SUMMARY/ABSTRACT An estimated 15?20 million people in the United States have peripheral artery disease (PAD). Despite advances in medical therapy, PAD remains associated with considerable cardiac and limb morbidity and mortality. Currently, more invasive procedures are performed in the lower extremities than in the heart, demonstrating increasing costs to the system of advanced PAD. Although the pathogenesis of coronary artery disease (CAD) is well characterized, the pathophysiology of PAD is less understood and the mechanism(s) that regulate this complex disorder remain uncertain. While antiplatelet therapy (as a class effect) decreases the incidence and complications from PAD, we noted that the effectiveness of antiplatelet therapy differs between PAD and other vascular phenotypes. In contrast to CAD, aspirin was not particularly effective in PAD nor was there clinical benefit with more potent P2Y12 inhibition. Clearly, new directions are needed to better understand the role of platelets in PAD pathogenesis and identify new therapeutic targets. Our group demonstrated the importance in coding and noncoding RNAs in regulating platelet activity. Leveraging our established cohort of PAD patients with well-phenotyped platelet activities, we demonstrated the importance of platelet?leukocyte interactions (in contrast to platelet?platelet aggregation) in the pathogenesis of PAD. Moreover, we identified an aberrant post-transcriptional regulation of platelets in PAD and demonstrated that platelets play a central effector role in activating monocytes and fostering inflammation in PAD. Here, we propose to comprehensively investigate the relationship between (1) platelet activity, (2) the platelet transcriptome, and (3) effector cell properties in patients with PAD. We will analyze stored platelet samples from >1,000 patients with longitudinal follow-up, many of whom provided serial collections. Leveraging these valuable platelet samples, we will focus on identifying novel platelet transcripts associated with vascular phenotypes and incident cardiovascular events. For example, we will compare patients with (1) PAD vs. other vascular phenotypes (e.g., CAD, carotid artery stenosis, abdominal aortic aneurysm), (2) stable PAD vs. CLI, and (3) incident cardiac (myocardial infarction) vs. limb (major amputation) events. Mechanistic studies using both cultured megakaryocytes and animal models with platelet-specific knock-in and knock-out of candidate genes will characterize how these processes are regulated. We are also well positioned to validate our findings in well-established local, national, and international cohorts. Our data suggest these types of studies can provide conceptual advances in our understanding of the mechanisms influencing the pathogenesis and severity of PAD. These insights could be leveraged to design clinical biomarkers and therapeutic strategies to treat and prevent vascular disease and its life-threatening complications.